Borrelia burgdorferi, the cauative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual genomic structure composed of a linear chromosome and a large number of linear and circular plasmids. Abundant evidence indicates that plasmid-encoded genes are critical for adaptation in the infectious cycle. In contrast to their essential role in vivo, plasmids are often lost during in vitro cultivation of B. burgdorferi. A major focus of our research is to determine the contributions of individual B. burgdorferi genes at each stage of the infectious cycle through a molecular genetic approach. Loss of plasmids in the outgrowth of a putatively isogenic clone presents a problem for subsequent genetic manipulations. We have investigated plasmid stability during in vitro propagation of B. burgdorferi. The impetus for these studies was to determine the feasibility of genetic manipulation of B. burgdorferi through a systematic assessment of the extent to which heterogeneity arose within a population during growth of cultures. We determined that genomic instability that arises during in vitro propagation of B. burgdorferi does not present an insurmountable obstacle to genetic studies designed to investigate the roles of specific genes in this pathogenic bacterium. We conclude that it is possible to generate isogenic wild type and mutant clones. Despite the large number of plasmids and their significance to the life cycle of B. burgdorferi, relatively little is known about the mechanisms of plasmid maintenance in borreliae. Previous work from our lab characterized the regions of circular plasmids that confer autonomous replication in B. burgdorferi, but the genetic elements responsible for linear plasmid replication had not been experimentally identified. To address this question, we constructed vectors from linear plasmids that replicate autonomously in B. burgdorferi. These vectors identify internal regions of linear plasmids necessary and sufficient for autonomous replication in B. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form in B. burgdorferi, indicating that plasmid maintenance functions are conserved. These shuttle vectors provide the tools necessary to dissect the components and functions of linear plasmid maintenance in B. burgdorferi. Antibiotic resistance genes allowing for efficient selection of mutants are an important prerequisite for genetic studies in B. burgdorferi. We previously constructed a kanamycin resistance cassette that greatly facilitated targeted mutagenesis in B. burgdorferi and the development of the first borrelial shuttle vector. Additional selectable markers are needed for creation of multiple mutations and complementation of B. burgdorferi mutants. To satisfy this need, we employed two different strategies to develop new antibiotic resistance cassettes for B. burgdorferi. These additional cassettes improve our repertoire of genetic tools and will facilitate molecular genetic investigations of the Lyme disease spirochete. In addition, our work may suggest strategies and aid in the development of tools for other bacteria that currently are not amenable to routine genetic manipulation. We recently succeeded in genetically manipulating a pathogenic B. burgdorferi clone and have undertaken comparisons of a series of mutant, complemented and wild type clones in the mouse-tick transmission cycle. These studies permit, for the first time, analysis of the contributions of individual gene products to infectivity or transmission at each stage of the infectious cycle. In collaboration with a consortium of extramural investigators, we also developed a membrane-based macro-array of all B. burgdorferi genes. This was used to identify significant temperature-induced changes in gene expression reflective of growth in either the tick vector or the mammalian host. Recently, in collaboration with Drs. Schwan and Gherardini at RML, we developed an oligo-based microarray on a glass slide for B. burgdorferi. We will use this oligo array to compare global patterns of gene expression between isogenic mutant and wild type organisms.